Date of Award

January 2016

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

David A. Hafler

Abstract

Immunotherapies targeting immune checkpoint receptors have shown remarkable efficacy in treating cancer. In the past couple years, two monoclonal antibodies targeting the checkpoint receptor PD-1 were approved by the FDA for treatment of advanced cancer; in clinical trials, these drugs induced deep and durable remissions, but also autoimmune side effects. Despite major advances, our understanding of checkpoint receptors and their roles in controlling autoimmune and anti-tumor immune responses remains limited. CD4+CD25highFoxP3+ regulatory T cells (Tregs) are critical regulators of immune responses in autoimmunity and malignancy, but the functional status of human Tregs expressing PD-1 remains unclear. TIGIT is a newly identified checkpoint receptor that inhibits T cell function by preventing assembly of the co-stimulatory receptor CD226 and by competing for their common ligand, CD155. Overexpression of TIGIT and CD155 has been reported in multiple cancers, while genetic studies link CD226 signaling to risk of developing multiple sclerosis and other autoimmune diseases. In order to elucidate the effect of PD-1 expression on Tregs, we examined functional and molecular features of PD-1high Tregs in healthy subjects and patients with glioblastoma, combining functional assays, RNA-sequencing, and cytometry by time of flight (CyTOF). In both glioblastoma and healthy subjects, circulating PD-1high Tregs displayed molecular signatures of exhaustion, reduced suppression of CD4 effector T cells, and production of IFNγ. Transcriptional profiling of tumor-resident Tregs revealed several genes co-expressed with PD-1 and associated with IFNγ production and exhaustion, as well as enrichment in exhaustion signatures compared to circulating PD-1high Tregs. CyTOF analysis on circulating and tumor-infiltrating Tregs from patients with glioblastoma treated with PD-1-blocking antibodies revealed that treatment shifts the profile of circulating Tregs towards a more exhausted phenotype reminiscent of the one of tumor-infiltrating Tregs, further increasing IFNγ production. In order to establish whether the TIGIT/CD226 axis contributes to the divergent T cell responses seen in cancer and autoimmunity, we used flow cytometry and immunohistochemistry to examine expression of these receptors on lymphocytes in multiple sclerosis and glioblastoma, central nervous system (CNS) diseases that appear to represent opposite ends of an immune regulatory spectrum; loss of immune regulation with activation of myelin-specific T cells leads to autoimmunity in multiple sclerosis, while impaired T cell responses in glioblastoma prevent tumor clearance. We found that TIGIT was highly expressed on glioblastoma-infiltrating CD8 T cells, but was near-absent from multiple sclerosis lesions. Similarly, TIGIT was significantly upregulated in circulating lymphocytes of patients with glioblastoma as compared to multiple sclerosis and healthy controls. In summary, high PD-1 expression on human Tregs identifies dysfunctional, exhausted Tregs that exist in healthy individuals and are enriched in tumor infiltrates, possibly losing function as they attempt to modulate the anti-tumor immune responses. Furthermore, TIGIT is differentially expressed on T cells in glioblastoma and multiple sclerosis, suggesting that it may have a crucial role in regulating CNS T cell immune responses and may thus be a target of therapeutic manipulation.

Comments

This thesis is restricted to Yale network users only. This thesis is permanently embargoed from public release.

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